The preparation of megaporous bodies, with potential applications in biotechnology, was attempted by following several strategies. As a first step, naive and robust scaffolds were produced by polymerization of selected monomers in the presence of a highly soluble cross-linker agent. Ion-exchange function was incorporated by particle embedding, direct chemical synthesis, or radiation-induced grafting. The total ionic capacity of such systems was 1.5 mmol H(+)/g, 1.4 mmol H(+)/g, and 17 mmol H(+)/g, respectively. These values were in agreement with the ability to bind model proteins: observed dynamic binding capacity at 50% breakthrough was ≅7.2 mg bovine serum albumin/g, ≅7.4 hen egg-white lysozyme (HEWL) mg/g, and ≅108 HEWL mg/g. In the later case, total (static) binding capacity reached 220 mg/g. It was observed that the structure and size of the megapores remained unaffected by the grafting procedure which, however, allowed for the highest protein binding capacity. Lysozyme supported on grafted body showed extensive clarification activity against a Micrococcus lysodekticus suspension in the flow-through mode, i.e., 90% destruction of suspended microbial cells was obtained with a residence time ≈ 18 min. Both protein capture and biocatalysis applications are conceivable with the 3D-megaporous materials described in this work.
Protein adsorption onto hydrophobic chromatographic supports has been investigated using a colloid theory surface energetics approach. The surface properties of commercially available chromatographic beads, Toyopearl Phenyl 650-C, and Toyopearl Butyl 650-C, have been experimentally determined by contact angle and zeta potential measurements. The adsorption characteristics of these beads, which bear the same backbone matrix but harbor different ligands, have been studied toward selected model proteins, in the hydrated as well as dehydrated state. There were two prominent groups of proteins observed with respect to the chromatographic supports presented in this work: loosely retained proteins, which were expected to have lower average interaction energies, and the strongly retained proteins, which were expected to have higher average interaction energies. Results were also compared and contrasted with calculations derived from adsorbent surface energies determined by inverse liquid chromatography. These results showed a good qualitative agreement, and the interaction energy minima obtained from these extended Derjaguin, Landau, Verwey and Overbeek calculations were shown to correlate well with the experimentally determined adsorption behavior of each protein.
Protein adsorption onto hydrophobic interaction chromatography supports was studied by a surface-thermodynamics approach. To gather relevant experimental information, contact angle measurements and zeta potential determinations were performed on three different commercial adsorbent beads, Phenyl Sepharose 6 Fast Flow, Toyopearl Phenyl 650-C and Source 15 Phenyl, having soft to rigid backbone structure. Similar information was obtained for a collection of model proteins, lysozyme, bovine serum albumin (BSA), polygalacturonase, aminopeptidase, chymosin, aspartic protease, beta-galactosidase, human immunoglobulin G, and lactoferrin, were evaluated in the hydrated and in the dehydrated state. Based on the mentioned experimental data, calculations were performed to obtain the (interfacial) energy versus distance profiles of nine individual (model) proteins on (commercial) beads of three different types. All of these beads harbored the phenyl-ligand onto a matrix of differentiated chemical nature. Extended Derjaguin, Landau, Verwey, and Overbeek (DLVO) calculations were correlated with actual chromatographic behavior. Typical chromatography conditions were employed. The population of model proteins utilized in this study could be segregated into two groups, according to the minimum values observed for the resulting interaction energy pockets and the corresponding retention volumes (or times) during chromatography. Moreover, trends were also identified as a function of the type of adsorbent bead under consideration. This has revealed the influence of the physicochemical nature of the bead structure on the adsorption process and consequently, on the expected separation behavior.
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